KR100279227B1 - Microbiological Preparation of Aromatic Hydroxyheterocyclic Carboxylic Acids - Google Patents

Abstract

A novel microbiological process for the preparation of hydroxylated heterocyclic carboxylic acids of the formula <IMAGE> starts from the corresponding heterocyclic carboxylic acid. This process is carried out in such a way that a) aerobic biomass which utilises nicotinic acid is cultivated in a molar ratio of nicotinic acid to mineral acid of 1 to 8, this ratio being ensured throughout the culture period, and then b) the hydroxylation of the corresponding heterocyclic carboxylic acid of the general formula. <IMAGE> in which R1 and X have the said meaning, is carried out with this biomass. Under these conditions it is possible in the culture period to accumulate microorganisms of the species Pseudomonas acidovorans DSM 7205, of the species Pseudomonas acidovorans DSM 7203, of the species Alcaligenes faecalis DSM 7204 and microorganisms with the identifier DSM 7202.

Description

Microbiological Preparation of Aromatic Hydroxy Heterocyclic Carboxylic Acids

The present invention provides a novel microbiological process for preparing hydroxy heterocyclic carboxylic acids of the general formula (I) from suitable heterocyclic carboxylic acids or their soluble salts using aerobic biomass using nicotinic acid or soluble salts thereof. Relates to a method of manufacture:

In the above formula,

R ₁ is a hydrogen atom or a halogen atom,

X is a nitrogen atom or CR ₂ -functional group, wherein

R ₂ is a hydrogen atom or a halogen atom.

The term nicotinic acid hereafter means not only their soluble salts but also their water soluble salts, such as sodium nicotinate, which is an alkali salt of nicotinic acid.

These hydroxy heterocyclic carboxylic acids are important intermediates for the preparation of pharmaceuticals. 6-hydroxynicotinic acid, for example, is an important intermediate for preparing 5,6-dichloronicotinic acid (Swiss Federal Patent No. 664 754) and is also a starting material for pharmaceutical actives (Setcliff et al., J. of Chem. And Eng. Data). , (1976), Vol. 21, Nr. 2, S.246).

A known example of a microbiological method of hydroxylating nicotinic acid to produce 6-hydroxynicotinic acid is described in EP 152 948. This method is first performed by selecting a nicotinic acid extract concentration at which the breakdown of nicotinic acid to 6-hydroxynicotinic acid is stopped in order to actually grow the microorganisms using nicotinic acid in the presence of yeast extract.

The method has the disadvantage that if the microorganism is grown using nicotinic acid in the presence of yeast extract, the cell-free fermentation solution is infected with the enzyme extract after growth or transformation, which causes the infection of the isolated 6-hydroxynicotinic acid.

Another disadvantage is that the method is carried out with a homogenous (microbiologically pure) microbial culture, which is particularly infected in large quantities during fermentation.

EP 92110425.3 describes a microbiological method for preparing 5-hydroxypyrazinecarboxylic acid from pyrazinecarboxylic acid.

The method uses an alkali salt of nicotinic acid to grow a uniform (microbiologically pure) microbial culture before the actual hydroxylation.

The method likewise has the disadvantage of carrying out the method with a homogeneous (microbiologically pure) microbial culture which is infected in large quantities during fermentation.

An object of the present invention is to solve the above-mentioned drawbacks and to provide a simple and ecological microbiological method for preparing hydroxyheterocyclic carboxylic acid of general formula (I).

The problem is solved by a novel method according to claim 1.

The method of the present invention,

a) growing aerobic biomass using nicotinic acid by using nicotinic acid and inorganic acid having a molar ratio of nicotinic acid to inorganic acid of 1: 8 and maintaining the ratio at all growth stages, and b) heterocyclic phase of general formula (II) The carboxylic acids or their soluble salts can be subjected to hydroxylation using the biomass to yield the desired hydroxylation final product of general formula (I):

In the above formula,

R ₁ and X are as defined above.

The term "aerobic biomass growth using nicotinic acid or salts thereof" then means:

The above-mentioned molar ratios of nicotinic acid-inorganic acid are inoculated and cultured in a precipitate under aerobic conditions to obtain aerobic biomass using nicotinic acid, that is, a carbon-, nitrogen- and energy source containing nicotinic acid and growing in the presence of oxygen.

As inoculum, soil specimens from different countries can be used, such as soils in municipal parks in Santander, Spain or vineyard soils in Pyrsperminen, next to the Swiss Federal Pepe.

The difference of the method according to the invention belonging to the above-mentioned method is that it is carried out using biomass existing from the mixed culture without using a uniform (microbiologically pure) microbial culture.

The molar ratio of nicotinic acid to inorganic acid, ie the addition of a mixture of nicotinic acid and inorganic acid for cell suspension, is carried out so that the molar ratio of nicotinic acid to inorganic acid is maintained at 1: 8 at all stages of growth.

As the inorganic acid, sulfuric acid, hydrochloric acid, nitric acid or phosphoric acid is used, for example, sulfuric acid is preferable.

While the biomass is growing (step a) it is advantageous to carry out the mixture addition using 3 to 5 moles of nicotinic acid per mole of sulfuric acid so that the molar ratio of nicotinic acid to sulfuric acid is guaranteed to be 3: 5. Preference is given to using from 4 to 5 moles of nicotinic acid per mole of sulfuric acid for growth.

Growth of aerobic biomass using nicotinic acid is typically carried out in inorganic salt medium, preferably in inorganic salt medium having the composition listed in Table 1 below.

The growth of the biomass is preferably carried out at pH 5-9, preferably pH 6-8.

The temperature during the biomass growth is effective between 15 and 50 ° C., preferably between 25 and 40 ° C.

Growth of biomass is typically carried out for 0.5 hours to 3 days.

Microorganisms of Pseudomonas acidovorans species DSM 7205, or Pseudomonas acidovorans species DSM 7203, or Algenogenes faecalis species DSM 7204 or DSM 7202 under these conditions at the growth stage. Or a mixture of these is appropriately enriched.

The microorganisms DSM 7205, DSM 7203, DSM 7204 and DSM 7202 were deposited on August 13, 1992 in Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH, Brown Schwede de-3300, Maschöderbeck 1-Be, under the Treaty of Budapest. .

These microorganisms are not known in the literature and are therefore also a component of the present invention. DSM 7202 microorganisms are in a taxonomic sense and not in a consensus or collateral sense.

The taxonomic characteristics of the microorganisms Pseudomonas acidovorans DSM 7205, DSM 7203 and Alcaligenes faecalis DSM 7204 are described below.

Taxonomic description of Pseudomonas acidovorans DSM 7205

Characteristic of this species

Cell form bacilli

Width μm 0.8 to 0.9

Length μm 1.5 to 9.0

Mobility +

Flagella polarity 1

Gram-response-

Lysis with 3% KOH +

Aminopeptidase (Cerny) +

Spores-

Oxidase +

Catalase +

growth

Anaerobic-

37/41 ° C +/-

pH 5.7-

Mac-Conkey's Agar +

SS-Agar +

Setlimide-Agar +

Testosterone-

Pigment

Not spread-

Diffused-

Fluorescent-

Piocyanine-

Acid generation (OF-test)

Glucose Aerobic?

Glucose anaerobic-

Glucose alkaline +

Gas generation from glucose-

Acid generation

From glucose-

From fructose +

From xylose-

From Adonitro-

From L-Arabinose-

From cellobiose-

From the sheet

From glycerol +

from m-inoseat-

From lactose-

From maltose-

From Raffinose-

From L-Rhamnose-

From the salinity-

From D-Sorbit

From Saccharos-

From trehalose-

From ethanol +

From the sheet

ONPG / PNPG-

ADH-

VP-

Indole-

NO ₂ generation from NO ₃ +

Phenylalanides aminase W

Levan formation from saccharose-

Recitase-

Urease-

Hydrolysis

Starch-

Gelatin-

Casein W

DNA-

Twin 80+

Asculin-

PHB-

Tyrosine decomposition +

Substrate availability

Acetate +

Adipate +

Caprate +

Citrate +

Glycolate +

Levulinate +

Maleate +

Malonate +

Phenyl Acetate +

L-Arabinose-

Fructose +

Glucose-

Mannose-

Maltose-

D-Xylose-

Mannitol +

Gluconate +

2-ketogluconate +

N-acetylglucosamine-

L-serine-

Quinate +

D, L-Tryptophan +

L-tartarate +

Acetamide +

α-aminobutyrate +

Ethanol W

Taxonomic description of Pseudomonas acidovorans DSM 7203

Characteristic of this species

Cell form bacilli

Width μm 0.8 to 1.0

Length μm 2.6 to 6.0

Mobility +

Flagella polarity 1

Gram-response-

Lysis with 3% KOH +

Aminopeptidase (Cerny) +

Spores-

Oxidase +

Catalase +

growth

Anaerobic-

37/41 ° C +/-

pH 5.7-

Mac-Conkey's Agar +

SS-Agar +

Setlimide-Agar +

Testosterone-

Pigment

Not spread-

Diffused-

Fluorescent-

Piocyanine-

Acid generation (OF-test)

Glucose Aerobic?

Glucose anaerobic-

Glucose alkaline +

Gas generation from glucose-

Acid generation

From glucose-

From fructose +

From xylose-

From Adonitro-

From L-Arabinose-

From cellobiose-

From the sheet

From glycerol +

from m-inoseat-

From lactose-

From maltose-

From Raffinose-

From L-Rhamnose-

From the salinity-

From D-Sorbit

From Saccharos-

From trehalose-

From ethanol-?

From the sheet

ONPG / PNPG-

ADH-

VP-

Indole-

NO ₂ generation from NO ₃ +

Phenylalanides aminase W

Levan formation from saccharose-

Recitase-

Urease-

Hydrolysis

Starch-

Gelatin-

Casein +

DNA-

Twin 80+

Asculin-

PHB-

Tyrosine decomposition +

Substrate availability

Acetate +

Adipate +

Caprate +

Citrate +

Glycolate +

Levulinate +

Maleate +

Malonate +

Phenyl Acetate +

L-Arabinose-

Fructose +

Glucose-

Mannose-

Maltose-

D-Xylose-

Mannitol +

Gluconate +

2-ketogluconate +

N-acetylglucosamine-

L-serine-

Quinate +

D, L-Tryptophan +

L-tartarate +

Acetamide +

α-aminobutyrate W

Ethanol +

Taxonomic description of Alcaligenes faecalis DSM 7204

Characteristic of this species

Cell form bacilli

Width μm 0.6 to 0.8

Length μm 1.0 to 2.0

Mobility +

Flagella

Gram-response-

Lysis with 3% KOH +

Aminopeptidase (Cerny) +

Oxidase +

Catalase +

growth

Anaerobic-

37/41 ° C +/-

pH 5.7-

Mac-Conkey's Agar +

SS-Agar +

Setlimide-Agar +

Pigment

Not spread-

Diffused-

Fluorescent-

Piocyanine-

Acid generation (OF-test)

Glucose Aerobic?

Glucose anaerobic-

Glucose alkaline +

Gas generation from glucose-

Acid generation

From D-glucose-

From D-fructose

From D-xylose-

ONPG / PNPG-

ADH-

VP-

Indole-

NO ₂ generation from NO ₃ +

Denitrogenation-

Phenylalanides aminase-

Levan formation from saccharose-

Recitase-

Urease-

Hydrolysis

Starch-

Gelatin-

Casein-

DNA-

Tween 80-

Asculin-

Tyrosine decomposition +

Substrate availability

Acetate +

Adipate-

Caprate +

Citrate +

Glycolate +

Revulinate-

D-maleate +

Malonate +

Phenyl Acetate +

L-Arabinose-

D-Fructose-

D-glucose-

D-Mannose-

Maltose-

D-Xylose-

Mannit-

Gluconate-

2-ketogluconate +

N-acetylglucosamine-

L-serine-

By hydroxylation hereafter is meant the hydroxylation of a heterocyclic carboxylic acid (substrate) and salts thereof, such as water soluble alkali salts.

The biomass is grown for actual transformation (hydroxylation) and then separated in the usual manner or added to the biomass directly grown heterocyclic carboxylic acid [formula (II)] for hydroxylation. do.

The actual hydroxylation of heterocyclic carboxylic acids (substrates) is carried out in the usual manner by those skilled in the art using cells that are not growing.

The actual hydroxylation of heterocyclic carboxylic acids was carried out at the growth stage of Pseudomonas acidovorans species DSM 7205, Pseudomonas acidovorans species DSM 7203, Algengenes faecalis It is carried out using microorganisms of species DSM 7204 or DSM 7202 or mixtures thereof.

Nicotinic acid, pyrazinecarboxylic acid or halogenated derivatives thereof can be used as the substrate. As the halogenated derivative of nicotinic acid or pyrazinecarboxylic acid, 5-chloronicotinic acid, 4-chloronicotinic acid or 6-chloropyrazinecarboxylic acid can be used. Preference is given to hydroxylating nicotinic acid with 6-hydroxynicotinic acid or pyrazinecarboxylic acid with 5-hydroxypyrazinecarboxylic acid.

The substrate for transformation can be added continuously or discontinuously. Substrate addition is suitably not more than 20% by weight, preferably 15% by weight of the substrate in the fermentor.

As the medium for carrying out the hydroxylation reaction, those conventional to those skilled in the art can be used, and preferably, the inorganic salt medium shown in Table 1 or the A-N medium shown in Table 4 is used.

Transformation is typically performed using cells having an optical density at 500 nm (OD ₅₅₀ ) or an optical density at 650 nm (OD ₅₅₀ ) of 5 to 100.

The transformation is suitably carried out at a temperature of pH 5 to 9, preferably 6.5 to 7.5 and 15 to 50 ° C, preferably 25 to 35 ° C.

After a typical conversion period of 5 to 24 hours, the hydroxylated heterocyclic carboxylic acid according to formula (I) is acidified with a cell-free fermentation broth or precipitated in the form of a salt that is difficult to dissolve in the usual manner. Isolate.

In particular, 6-hydroxynicotinic acid or 5-hydroxypyrazine carboxylic acid is isolated as hydroxylated heterocyclic carboxylic acid.

Example 1

(a) Growth of biomass

The fermentation is carried out using 1 g of nicotinic acid per liter at a pH of 7.0 and 30 ° C. and an aeration rate of 5 to 20 liters / minute in a non-sterile inorganic salt medium (Table 1) in a 15 liter working volume fermentor. To adjust the pH, acid in the form of an aqueous suspension of 307 g nicotinic acid (2.5 moles), 49 g (0.5 moles) H ₂ SO ₄ and 1 liter of water was applied to the air pressure from a vessel equipped with a stirrer, fixed to the cover of the fermentor. It is added to the medium through a ball valve controlled by. Inoculate the fermenter with 500 ml of sediment from the Swiss Federal Pepe water purification plant (Table 2). After 36 hours the fermentor is emptied to 1 liter and filled with fresh medium. The procedure is repeated after 24 hours, 48 hours and 72 hours.

b) hydroxylation (preparation of 6-hydroxynicotinic acid)

Once an optical density of 5-20 at 550 nm is obtained, biomass is added to determine the growth rate of specific 6-hydroxynicotinic acid with a spectrophotometer. For this the biomass is first washed once with 0.9% (w / v) NaCl solution. Then a quartz vessel preheated to 30 ° C. containing 6.5 g of nicotinic acid / liter, 10.1 g of KH ₂ PO ₄ / liter and 4.0 g of K ₂ HPO ₄ / liter, 2990 μl of a solution of pH 7.0 (1 10 μl of the cell suspension is added. The absorbance of the vessel is then measured at 550 nm and then the linear increase in absorption is calculated at 295 nm per minute from the same vessel.

Inactivity (U) is determined according to the following equation:

The fermentation is repeated with the Swiss federation, the sediment from the Zermatt water purification plant, the soil from the Piersterminen region and the soil from the Lac de Joux region and the soil from the Spanish Santander region (Table 2).

Example 2

Preparation of 5-hydroxypyrazinecarboxylic acid

Centrifuge biomass from Presentations 4, 5 and 5 (Table 2) and wash once with 0.9% (w / v) NaCl solution. The cells are appropriately resuspended in 1 liter solution containing 0.5 mole (70 g) of pyrazinecarboxylic acid-ammonium salt (pH 7.0). The optical density is 20 at 650 nm. After 16 hours of incubation under aerobic conditions at a temperature of pH 7.0 and 30 ° C., quantitative conversion of pyrazinecarboxylic acid to 5-hydroxypyrazinecarboxylic acid is measured using a UV spectrometer. Formation of 5-hydroxypyrazinecarboxylic acid is carried out using microorganisms that are not at rest.

For control, Pseudomonas acidoborance D3 (DSM 4746), which is particularly suitable for the industrial preparation of 5-hydroxypyrazine, is grown as described above and then the inversion reaction is carried out by the method described in EP 92110425.3. The results are listed in Table 2 below.

Table 1: Composition of Medium of Inorganic Salts

MgCl ₂ .6H ₂ O 0.8 g / L

CaCl ₂ 0.16 g / L

Na ₂ SO ₄ 0.25 g / ℓ

K ₃ PO ₄ .2H ₂ O 0.7 g / ℓ

Na ₂ PO ₄ .12 H ₂ O 2.4 g / ℓ

SLF 1.0 g / ℓ

FeFDTA 15.0 g / ℓ

The composition of trace elements (SLF) in the inorganic salt medium is as follows;

KOH 15.0 g / ℓ

EDTANa ₂ .2H ₂ O 100.0 g / l

ZnSO ₄ .7H ₂ O 9.0 g / ℓ

MnCl ₂ .4H ₂ O 4.0 g / l

H ₃ BO ₃ 2.7 g / ℓ

CoCl ₂ .6H ₂ O 1.8 g / ℓ

CuCl ₂ .2H ₂ O 1.5 g / l

NiCl ₂ .6H ₂ O 0.18 g / l

Na ₂ MoO ₄ .2H ₂ O 0.2 g / ℓ

The composition of FeEDTA is as follows:

EDTA Na ₂ .2H ₂ O 5 g / l

FeSO ₄ .7H ₂ O 2 g / ℓ

(After adding all, the solution has a pH of 7.0).

TABLE 2

Destination:

(1) Sediment from the water purification plant in Fa.LONZA, Switzerland

(2) River soils in the Lac de Joux, Le Sentier, Swiss Confederation

(3) Sediments from water purification facilities in Zermatt, Switzerland

(4) Soils in the vineyards of the Piersferminen region in Pisp, Switzerland

(5) Soil in municipal parks in Santander, Spain

[Examples 3 to 6]

The biomass grown according to Example 1a) may be pure microorganisms as follows:

Pseudomonas acidovorans DSM 7205

Pseudomonas acidovorans DSM 7203

Alcaligenes faecalis DSM 7204

. Microorganisms in the DSM 7202

These microorganisms are grown under the following conditions and added to hydroxylate nicotinic acid with 6-hydroxynicotinic acid.

The results are shown in Table 3 below. The microorganisms are grown using 2 g of sodium nicotinate per liter at 30 ° C. and pH 7.0 in 7 liters containing 5 liters of AN medium (Table 4). 5N NaOH and 8.5% (v / v) H ₃ PO ₄ are used to adjust the pH. After 18 hours of growth, 2 g of sodium nicotinate per liter is added to the fermentation broth. If cells in exponential growth phase are found, fermentation is stopped and microorganisms are separated from the medium by centrifugation. The cells are appropriately suspended in 500 ml solution containing 0.27 mole (40 g) sodium nicotinate (pH 7.0). The optical density is 20. The hydroxylation of nicotinic acid with 6-hydroxynicotinic acid is observed by spectrophotometer (Table 3).

TABLE 3

Table 4: A-N Medium

(The pH of the solution is 7.0.)

Claims (10)

(a) Pseudomonas acidoboranth using nicotinic acid or salts thereof and inorganic acids having a molar ratio of nicotinic acid or a soluble salt thereof to an inorganic acid of 1: 8 and maintained at the above ratio at all stages of growth, Pseudomonas acidovorans species DSM 7205 and / or Pseudomonas acidovorans species DSM 7203 and / or Alcaligenes faecalis species DSM 7204 and / or aerobic microorganisms selected from microorganisms of DSM 7202 And (b) microbiologically preparing the hydroxylated heterocyclic carboxylic acid of the following general formula (I) by hydroxylating the heterocyclic carboxylic acid of the following general formula (II) or a soluble salt thereof using the microorganism. How to: In the above formula, R ₁ is a hydrogen atom or a halogen atom, X is a nitrogen atom or CR ₂ -functional group, wherein R ₂ is a hydrogen atom or a halogen atom. The process of claim 1, wherein sulfuric acid is used as the inorganic acid in step (a) such that the molar ratio of nicotinic acid or soluble salts thereof to sulfuric acid is 3; 5. The method according to claim 1 or 2, and (b) hydroxyating the heterocyclic carboxylic acid nicotinic acid or a soluble salt thereof with 6-hydroxynicotinic acid. The process according to claim 1 or 2, wherein in step (b), pyrazinecarboxylic acid or a soluble salt thereof, which is a heterocyclic carboxylic acid, is hydroxylated with 5-hydroxyparazincarboxylic acid. The process according to claim 1 or 2, wherein the growth in step (a) and the hydroxylation in step (b) are carried out at a temperature of 15 to 50 ° C and a pH of 5 to 9. Microorganism of Pseudomonas acidovorans species DSM 7205 using nicotinic acid or soluble salts thereof. Pseudomonas acidovorans species DSM 7205 and / or Pseudomonas acidovorans species DSM 7203 and / or Alcaligenes faecalis species using nicotinic acid or soluble salts thereof Or hydroxylation of nicotinic acid using a microorganism of DSM 7202. Microorganism of Pseudomonas acidovorans species DSM 7203 using nicotinic acid or soluble salts thereof. Microorganism of Alcaligenes faecalis species DSM 7204 using nicotinic acid or soluble salts thereof. Microorganisms of Alcaligenes faecalis species DSM 7202 using nicotinic acid or soluble salts thereof.